In the first two funding cycles of this grant, we have characterized the molecular structure and function of Cutaneous Lymphocyte Antigen or CLA. We have also made the surprising observation that in humans, the vast majority (>90 percent) of CLA+ skin homing T cells are not in peripheral blood, but rather are in skin at any given time. These cells appear to enter skin directly from blood, through constitutively expressed E selectin, chemokine ligands, and LFA-1 on dermal microvasculature. These observations suggest that memory responses in skin to antigens originally encountered through skin may not require acute extravasation of circulating memory T cells. To more fully define the immunophysiology of these phenomena, we have used murine models of cutaneous infection with vaccinia virus to track a T cell mediated, antigen-specific immune response from draining lymph node and subsequently to both skin as well as other peripheral tissues and secondary lymphoid tissues. These observations provided us with a model of how cutaneous immunization leads to both a robust skin homing response via imprinting of effector memory T cells in draining lymph node, as well as a vigorous systemic response, by migration of subset of central memory T cells throughout the body. In the next five years, we propose to further dissect antigen specific T cell trafficking after cutaneous infection as means to better understand the biology of skin homing T cell trafficking and recirculation through skin. Using gene deficient mice, we will explore the role of the skin homing molecules that discovered were important in the last grant cycle, including PSGL-1, CD43, and FucTVII. We will use parabiotic mouse models to test hypotheses about the trafficking of T cells from skin to LN, blood, and ultimately back to skin. Finally, we will use these models to discover mechanisms and the kinetics of T cell migration through skin. Taken together, these studies will allow us to test clinically important hypotheses about the biology of skin homing T cells. Understanding the biology of these cells is central to modifying their function in the course of treating human diseases. Moreover, because many vaccines are delivered through skin, understanding how such cutaneous responses lead to systemic immunity will help us improve the quality and impact of modern vaccine therapy. PROJECT NARRATIVE: A better understanding of how skin homing T cells are generated after skin immunization, and how memory T cells enter and exit skin, is critical for developing better treatments for skin diseases. It is also critical for development and optimization of vaccines, the majority of which are given via skin immunization. Knowledge generated from this grant application over the next five years will have a significant impact on both areas.